During fertilization in mammals, the sperm utilizes repetitive calcium signals to activate the oocyte's developmental program. The fertilizing sperm triggers a release of calcium from the intracellular stores that is followed by a calcium influx through channels in the oocyte plasma membrane. Although a calcium influx generated by store depletion is vital for the generation of the long-lasting signal, the mechanisms that regulate this capacitative calcium entry are largely unknown. The overall goal of the proposal is to define the regulatory mechanisms of capacitative calcium entry in porcine oocytes. Our aim is to investigate the role of STIM1 in capacitative calcium entry. In somatic cells, STIM1 has been suggested to serve as a calcium sensor to detect calcium levels in the intracellular stores and in preliminary experiments we demonstrated that porcine oocytes also express a STIM1 homolog. Here we propose to determine the dynamics of STIM1 gene expression during oocyte maturation by means of quantitative RT-PCR. By using a fluorescently labeled probe, STIM1 will be shown to translocate from the cytosol to the plasma membrane after store depletion and data will be presented that downregulation of its function with siRNAs inhibits capacitative calcium entry. The information obtained through the proposed experiments will help in the development of novel oocyte activation methods that will increase the efficiency of nuclear transfer technology. This is believed to be critical for the large scale production of transgenic large animals. Such animals with specific genetic modifications are regarded as extremely valuable models for a great number of human diseases: they help to better understand disease mechanisms and also, to develop new strategies for therapeutic interventions. The information obtained through the proposed experiments will contribute to the effective production of transgenic large animals to be used as models for human diseases. By using the animals with specific genetic alterations it will be possible to better understand the mechanism of diseases and develop new and effective therapies. [unreadable] [unreadable] [unreadable]